Quantitation by High Resolution Mass Spectrometry: Case Study of TOF MS for the Quantitation of Allopurinol from Human Plasma Shaokun Pang 1, Weixing Sun 2, Adrien Musuku 2, Xavier J. Misonne 1 1 SCIEX, Redwood City, CA; 2 Pharmascience Inc., Montreal, RUO-MKT-11-4490-A QC, Canada For Research Use Only. Not for use in diagnostic procedures.
Introduction Multiple reaction monitoring (MRM) methods has been the most popular tool in bioanalysis for the past two decades. Isobaric interferences present in complex matrices can be a challenge when they elute at or near the retention time of the analyte of interest in MRM chromatograms. The high resolution and mass accuracy on a Time-of-flight (TOF) MS provides better selectivity and can achieve higher signal-to-noise ratios (S/N) by filtering out interferences caused by isobaric compounds in the sample matrix. In addition to the targeted analyte signals, the rich TOF MS data can provide more information on matrix background, metabolites and interferences. This versatility provides users a valuable trouble-shooting tool and enables users to develop better methods 2 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Allopurinol Assay In this study, we demonstrate the versatility of TOF data acquisition using the SCIEX TripleTOF 5600+ system in an assay of allopurinol Allopurinol is a medication used primarily to treat excess uric acid in the blood and its complications, including chronic gout. It is a xanthine oxidase inhibitor and is administered orally. An allopurinol assay was previously established on a SCIEX API 5000 TM system and 2 isobaric peaks were present in the MRM chromatogram of the analyte * * * Allopurinol Allopurinol- 13 C, 15 N 2 3 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Allopurinol MRM Chromatograms Representative MRM chromatograms of blank plasma (A/B) and LLOQ (C/D) samples for Allopurinol 1.45 min (A and C) and its internal standard (B and D) 1.45 min. Allopurinol Allopurinol-IS Allopurinol Allopurinol-IS Isobaric endogenous compounds seen at 1.07 min and 2.43 min in the Allopurinol MRM chromatogram 4 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Calibration Curve and Statistics of MRM Quantitation Calibration curve linear Quantitation range was 10 to 3200 ng/ml QC Statistics are shown below Sample Name Actual Mean Calculated Concentration Concentration (ng/ml) %CV Accuracy LLOQ 10.0 10.0 4.77% 100.00% QC A 30.0 30.7 2.39% 102.33% QC B 1600 1584 2.52% 99.02% QC C 3200 3108 1.97% 97.13% 5 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Allopurinol TOF MS Chromatograms A different LC system was used for analysis on the SCIEX TripleTOF 5600+ system and retention times are shifted The peaks in the TOF MS chromatogram (A) were generated from an XIC (10 mda extraction width) of the parent ion m/z 135.031 and IS 138.029. Allopurinol elutes at 2.47 min (1.45 min in MRM) and the isobaric compounds at 1.45 min in the MRM at 2.21 min in the TOF MS chromatogram The isobaric compound at 2.43 min in the MRM chromatogram is not present in the TOF MS chromatogram A new compound at 1.12 min is revealed in the TOF MS XIC Allopurinol MRM TOF MS 6 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Allopurinol TOF MS Quantitation A calibration curve and QC samples were run on the SCIEX TripleTOF 5600+ system and the TOF MS XIC of Allopurinol and Allopurinol-IS were used to build the calibration curve Calibration Standard Statistics QC Sample Statistics Quantitation using TOF MS gave matching precision, accuracy, and linearity to the nominal resolution triple-quad MS. 7 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Allopurinol TOF Product Ion Chromatograms (MRM HR ) The peaks in the TOF product ion (MRM HR ) chromatogram (B) were generated from an XIC (10 mda extraction width) of the product ion m/z 92.026 Allopurinol elutes at 2.47 min (1.45 min in MRM) and the isobaric compounds at 2.21 and 3.15 minutes The new compound at 1.12 min revealed in the TOF MS XIC is not present in the MRM HR chromatogram Allopurinol MRM TOF Product Ion B Allopurinol 8 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Allopurinol MRM HR Quantitation The TOF Product Ion XIC (MRM HR ) of Allopurinol 92.026 and Allopurinol-IS were used to build the calibration curve Calibration Standard Statistics QC Sample Statistics The calibration curve for quantitation using TOF Product Ion XIC (MRM HR ) was concave in shape and required a polynomial fit The polynomial fit gave acceptable precision and accuracy to the TOF MS and nominal resolution triple-quad MS. 9 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
TOF MS Spectra of Allopurinol The 3.15 min isobaric peak (B) was eliminated from the TOFMS XIC due to its molecular ion (135.0456) larger-than ±0.005 Da (D) B The Allopurinol molecular ion has an m/z of 135.0309 (C). TOF MS Allopurinol TOF MS 10 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
TOF Product Ion Spectra of Allopurinol The compound at 1.12 min revealed in the TOF MS XIC (A) is not present in the MRM HR chromatogram because there is no product ion signal at 92.023 (E). TOF Product Ion 11 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
MRM HR Quantitation The MRM HR standard curve was concave in shape, which is characteristic of a constant systematic contribution of intensity (background) in the chromatogram In the background there is also a contribution of 135.0 92.026 as shown on left This background intensity in MRM HR affects the linearity of the calibration curve at low concentrations and results in the concave shape Background intensity in MRM HR (1.5 to 1.7 min) This low mass background contribution was not significant on the SCIEX API 5000 TM system 12 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Summary In this case study, we have demonstrated that the TOF MS instrument is able to conduct a bioanalytical assay with matching precision, accuracy, and linearity to a nominal resolution triple-quad MS. Analysts have a choice of using either MS or MS/MS data to quantify the analyte. TOF data (MS and MS/MS) will provide additional specificity in addition to the specificity from high resolution. Beyond the quantitation capability, and unlike MRM data, TOF data also provides more information during method development for users to trouble-shoot issues that could interfere with the method and/or the results 13 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.
Trademarks/Licensing AB Sciex is doing business as SCIEX. For Research Use Only. Not for use in diagnostic procedures. 2016 AB Sciex. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. 14 2016 AB Sciex RUO-MKT-11-4490-A For Research Use Only. Not for use in diagnostic procedures.